Researchers have identified a set of antibodies that they claim can be used to tag and remove undifferentiated human embryonic stem cells (hESCs) from differentiated cells in a population, potentially eliminating the risk of teratoma formation in future patients receiving hESC-derived cell therapies.

The antibodies, developed by a Stanford University School of Medicine team, includes one that they have called antistage-specific embryonic antigen (SSEA)-5, which binds to H type-1 glycan, a previously unidentified antigen they say is highly and specifically expressed on human pluripotent stem cells (hPSCs).

The potential for teratoma formation by residual undifferentiated cells is one of the major drawbacks to the clinical use of hPSC-derived cell therapies, Dr. Drukker et al. report. One potential resolution of this problem would be the removal of undifferentiated cells before transplantation. Prior research resulted in the development of a mAb that is capable of inducing cell death in pure cultures of undifferentiated hESCs. Its use, however, has not been extended to the depletion of residual teratoma-initiating cells from heterogenous differentiated cultures.

To try and generate a universally applicable protocol for removing residual and undifferentiated cells from heterogeneous populations, the Stanford team set out to identify surface-marker combinations that would allow separation of these cells using fluorescence-activated cell sorting (FACS). Their hunt involved two mAb sources: a mouse hybridoma library raised against undifferentiated hESCs and a library of commercially available mAbs.

One antibody identified in the hybridoma libary was found to highly label undifferentiated but not differentiated hESCs and was designated anti-SSEA-5. To test the antibody in vivo they dissociated hESC-derived teratomas to single cells and injected either SSEA-5+ or SSEA-5- cells in immunodeficient mice.

All seven SSEA-5+ transplants from three independent experiments formed large teratomas, whereas only 3 out of 11 SSEA-5- transplants gave rise to any growths, and these were small. In addition, the researchers found no evidence of SSEA-5 expression in any samples from a panel of 12 human tissues from 7-month-old fetuses.

Further analysis of the SSEA-5 antibody suggested it specifically binds to H type-1 glycan antigens on the surface of hESCs. Importantly, anti-SSEA-5 didn’t bind to any glycan without the H-1 motif, including both H type-2 (H-2) and globo-H antigens. Moreover, glycans bound by commercial mAbs against H-2, SSEA-3, and SSEA-4 also showed no overlap with those bound by anti-SSEA-5.

The H-1 glycan is modifyable to other glycans including Lewis and ABO glood group antigens. When the researchers looked closer at glycan antigens on hESC and human induced pluripotent stem cell (IPSC) lines, they found that as the cells start to differentiate, glycan expression shifts from type 1 to type 2. This, they point out, suggests that blood group antigens exhibiting a type-1 backbone (such as H-1) are specific to undifferentiated hPSCs and are potentially replaced with glycans exhibiting a type-2 backbone during the course of differentiation.

Importantly, in vivo assays confirmed that the SSEA-5 antibody could be used to remove undifferentiated stem cells from heterogeneous populations. In one experiment the team confirmed that mixtures of cells depleted of SSEA-5+ cells formed only small tumors and in only three out of eight replicates. In a second experiment they isolated cells that demonstrated either high SSEA-5 expression or low SSEA-5 expression. Teratomas formed from both these isolated populations, but those from the SSEA-5-high cells were initially significantly larger.

Although the SSEA-5 antibody was capable of removing most pluripotent stem cells from heterogeneous populations, it was not 100% effective. The researchers therefore moved on to identify additional markers that could be used alongside SSEA-5 to ensure complete removal of teratoma-initiating cells.

They analyzed a panel of commercially available mAbs and found five candidate trypsin-insensitive IPSC markers: CD9, CD30, CD50, CD90, and CD200. Of these, CD9, CD30, and CD90 have previously been shown to correlated with pluripotency.

To confirm that this set of five markers plus the SSEA-5 marker (collectively known as the pluripotency surface markers, or PSMs) could identify all undifferentiated cells, the team performed multicolor flow cytometry analysis, which showed that a single-cell population co-expressing high levels of four PSMs (CD9, CD50, CD90, and SSEA-5) decreased in proportion during differentiation from 52% to 6% at days 3 and 10 of retinoic acid treatment, respectively.

They then carried out a bioinformatics analysis to evaluate the specificity of PSM combinations for undifferentiated iPSCs. This involved stratifying > 27,000 human microarray samples, of which 120 samples represented pluripotent sources including hESCs, iPSCs, and germ cell tumors. Analyses showed that over 99% of the nonpluripotent tissues didn’t express high levels of CD9, CD30, CD90, and CD200, whereas almost all pluripotent samples did.

CD50 couldn’t be included in the analysis as probes were insensitive, and SSEA-5 is a glycan so also had to excluded. This specificity of marker expression was maintained when combinations of three PSMs were assessed, but declined with PSM pairs and even more with single PSMs.

Significantly, immunohistochemical analysis of 7-month-old human fetal tissues revealed that approximately half of the analyzed organs were labeled with three or more PSMs but that labeled structures within tissues rarely overlapped. “Taken together, these results suggest that concurrent high expression of three PSMs is rarely found in nonpluripotent tissues,” the authors write.

To functionally test whether three PSMs can distinguish and therefore eliminate undifferentiated from differentiated iPSCs, they sorted heterogeneously differentiated cultures using the representative mAb combinations. The results showed that SSEA-5/CD9/CD90-high population cells formed large teratomas with evidence of three germ layers, whereas the SSEA-5/CD9/CD90-low population didn’t.

Rather, all growths emerging from the SSEA-5/CD9/CD90-low population exhibited histologic evidence of only epithelium and mesenchyme and appeared to lack structures typical of teratomas. Similar results were obtained for cells sorted according to high or low SSEA-5/CD50/CD200 expression. In contrast, cells separated according to high or low expression of two classic iPSC markers, TRA-1-81 and SSEA-4, were all capable of forming teratomas consisting of tissues of the three germ layers.

“The limited tissue repertoires exhibited by the SSEA-5/CD9/CD90-low and SSEA-5/CD50/CD200-low grafts are not consistent with populations of pluripotent cells but rather of precursors committed to later developmental stages,” the team notes.” At this point, we are unaware of any clinical hurdles that may be imposed by such embryonic precursors.

“We demonstrate here immunodepletion using three surface-marker combinations to remove residual teratoma-initiating cells from heterogeneously differentiated hESC cultures,” they conclude. In particular, Dr. Tang points out, the results flag the importance of glycans in stem cell biology. “Many glycans are highly expressed in embryonic stem cels, but not in differentiated cells. This warrants further study and may lead to new understandings about embryonic stem cell biology.”

The researchers admit that the PSMs described in their published work won’t necessarily represent a complete list, and the identification of additional markers distinguishing pluripotent from differentiated cells will further help the task of depleting teratoma-initiation cells.

Nevertheless, they state, “It is our hope that antibodies against SSEA-5 and the additional PSMs would be immediately applied to advance hPSC research and to ensure the safety of patients undergoing clinical trials using hPSC derivatives.”

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